Adult-onset diabetes treatment method

ABSTRACT

Methods of inducing weight loss and treating adult-onset diabetes in a mammal in need thereof by administering to the mammal Momordica lectin or pokeweed mitogen chloroform precipitatable fraction. Lectin pharmaceutical compositions are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional Application of U.S. application Ser.No. 09/218,198, filed Dec. 22, 1998, now U.S. Pat. No. 6,235,286, andincorporated herein by reference in its entirety, which in turn claimspriority as a Continuation-in-Part of U.S. patent application Ser. No.08/799,094, filed Feb. 11, 1997, which issued as U.S. Pat. No. 5,851,531on Dec. 22, 1998, and in turn claims priority benefit of U.S.Provisional Application No. 60/017,179, filed May 9, 1996. Thedisclosure of U.S. Pat. No. 5,851,531 is incorporated herein byreference.

BACKGROUND OF THE INVENTION

The present invention relates to methods of treating adult onsetdiabetes by administering to a patient in need thereof an effectiveamount of a lectin capable of inducing a decline in food intake andinducing hypoglycemia. The present invention also relates to methods ofinducing weight loss by administering an effective amount of a lectincapable of inducing a decline in food intake and repeating theadministering step to maintain the decline in food intake. Inparticular, the present invention relates to methods of treatingadult-onset diabetes and inducing weight loss with the mixture oflectins known as pokeweed mitogen and the bitter pear melon lectin.

Adult-onset Type II diabetes mellitus is a major health problem in thiscountry. Because it is exacerbated by obesity, treatment of this diseaseis often two-pronged, using drugs to maintain normal blood glucoseconcentrations and diet therapy to promote weight loss. However, currenthypoglycemia drugs are not always effective, and adherence to diettherapy is generally poor.

Lectins are a general class of proteins that bind to carbohydrates. Thebinding of these lectins to carbohydrates that are part of cell membranereceptors can result in cell activation. This is particularly wellcharacterized with cells of the immune system. Lectins are widely usedto stimulate immune function in in vitro studies.

The crude extract of the lectins from the pokeweed plant, Phytolaccaamericana is referred to as pokeweed mitogen. Pokeweed mitogen is amixture of five lectins, which are designated as Pa-1 to Pa-S. Theserange in size from 19 to 30 kD, and contain approximately 3 percentcarbohydrate. The ligand for all the pokeweed lectins is N-acetylglucosamine. Despite binding to the same ligands, there is a disparitybetween the bioactivity of the five pokeweed mitogen lectins. Only Pa-1is a polymer, and only Pa-1 stimulates B cells. Further, Pa-2 to Pa-5,all of which stimulate T cells, do so with widely differing potency.

Effects attributed to pokeweed mitogen include stimulation of the immunesystem, with the appearance of plasma cells in the peripheralcirculation, the release of interferon-y and tryptophan metabolites, andtransient splenomegaly secondary to blood engorgement. Zazula et al.,P.S.E.B.M., 133, 1088-92 (1970), disclosed that the intravenousinjection of pokeweed mitogen to mice produced a marked increase in theweight of the spleen, with an increase in the number of nucleated cells,lymphoblasts, and mitotic figures. A marked increase in the total numberof plasma cells was also produced, but these cells did not appear in theperipheral blood, and the effect of pokeweed mitogen was limited to atransient leukopenia. One to three days after the administration ofpokeweed mitogen at a level of 175 mg/kg, all mice lost an average of1.5 g of body weight, but by day five showed a net weight gain. Weightloss following pokeweed mitogen infusion is not consistently reported inthe literature.

Wheat germ agglutinin is a lectin that shares structural and bindingproperties with pokeweed mitogen lectins. Mice given wheat germagglutinin developed a non-significant hypoglycemia and a decline infood intake, but no weight loss. Like Pa-2 to Pa-5, wheat germagglutinin stimulates T cells.

There exists a need for a pharmaceutical composition for the treatmentof diabetes mellitus that produces both a lowered circulating glucoselevel and an anorexic state that results in the loss of body weight.

SUMMARY OF THE INVENTION

This need is met by the present invention. It has now been discoveredthat a chloroform precipitatable fraction of pokeweed mitogen likely tobe lectin Pa-1 not only possesses the insulinomimetic properties ofwheat germ agglutinin, thereby producing a decline in circulatingglucose levels by binding to cell insulin receptors, but, unlike wheatgerm agglutinin, Pa-1 also binds to insulin receptors in the brain toproduce an anorexic state and consequent weight loss. It has furtherbeen discovered that the lectin from the bitter pear melon Momordicacharantia also induces hypoglycemia and an anorexic state andconsequential weight loss to a degree equivalent to that induced by thePa-1 chloroform precipitate.

Therefore, according to one aspect of the present invention, there isprovided a method of treating adult-onset diabetes in a mammal in needthereof by administering to the mammal a pokeweed mitogen chloroformprecipitatable fraction in an amount between about 1 and about 100micrograms per kilogram of body weight that is effective to inducehypoglycemia in said mammal.

According to another aspect of the present invention there is provided amethod of treating adult-onset diabetes in a mammal in need thereof byadministering to the mammal a dosage form consisting essentially ofMomordica lectin in an amount effective to induce hypoglycemia in themammal. Preferred methods in accordance with this aspect of the presentinvention further include the step of continuing the administering stepto maintain the hypoglycemia state. The preferred dosage amount isbetween about 1 and about 100 micrograms per kg of body weight per day,and even more preferable is an amount effective to induce a decline infood intake. An even more preferred method continues the administeringstep daily until the decline in food intake produces a weight loss of atleast about 10 percent.

The lectins of the present invention also induce a decline in foodintake and consequential weight loss and may be administeredadvantageously to any patient who would obtain a health benefit from aloss of weight. Therefore, according to another aspect of the presentinvention, there is provided a method of inducing weight loss in amammal in need thereof by administering to the mammal a lectin selectedfrom a pokeweed mitogen chloroform precipitatable fraction and Momordicalectin in an amount between about 1 and about 100 micrograms per kg. ofbody weight per day that is effective to stimulate a decline in foodintake in the mammal, and continuing the administering step to maintainthe decline in food intake.

The present invention incorporates the discovery that the lectins of thepresent invention may be administered in an amount that stimulates adecline in food intake without inducing hypoglycemia. Therefore,preferred weight loss methods according to the present inventionadminister to the mammal a pokeweed mitogen chloroform precipitatablefraction or Momordica lectin in an amount effective to induce a declinein food intake in the mammal without inducing hypoglycemia.

However, dosage levels that induce both a decline in food intake andhypoglycemia may be advantageously employed in the treatment ofadult-onset diabetes to control both blood sugar levels and patientweight. Therefore, according to another aspect of the present invention,there is provided a method of treating adult-onset diabetes in a mammalin need thereof by administering to the mammal a dosage form consistingessentially of Momordica lectin in an amount effective to induce adecline in food intake and induce hypoglycemia in the mammal, andrepeating the administering step daily until the decline in food intakeproduces a weight loss of at least about 10%.

The present invention also includes pharmaceutical compositionsconsisting essentially of an effective amount of lectin selected frompokeweed mitogen chloroform precipitatable fraction and Momordicalectin, and a pharmaceutically acceptable carrier. The pharmaceuticalcompositions of the present invention may be in either oral or systemicdosage forms.

While not being bound by any particular theory, it is believed thatpokeweed mitogen chloroform precipitatable fraction and Momordica lectinproduced weight loss in addition to hypoglycemia by crossing theblood-brain barrier and binding to insulin receptors in the brain. It isbelieved that other lectins that produced only hypoglycemia without acorresponding weight loss are capable of binding to cell membraneinsulin receptors to increase cellular glucose uptake and reducecirculating glucose levels, but are incapable of crossing theblood-brain barrier to bind to insulin receptors in the brain.

While weight loss following pokeweed mitogen infusion was notconsistently shown in the prior art literature, the contradictoryfindings are believed to have resulted from differing preparations ofthe pokeweed mitogen, with the predominance of different forms of thelectins. The present invention incorporates the discovery that in orderto obtain consistent weight loss, a pokeweed mitogen chloroformprecipitatable fraction must be employed.

The above and other objects, features and advantages of the presentinvention will become clear from the following description of thepreferred embodiments considered in conjunction with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a depicts the effect of pokeweed mitogen on circulating glucoseand lactate in mice;

FIG. 1b depicts the effect of pokeweed mitogen on circulating insulin inmice;

FIG. 2a depicts the effect of pokeweed mitogen on circulating glucoseand food intake in mice;

FIG. 2b depicts the effect of pokeweed mitogen on body, carcass andepididymal fat pad weight in mice;

FIG. 3a depicts the effect of pokeweed mitogen on body weight in mice;and

FIG. 3b depicts the effect of pokeweed mitogen on food intake in mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

It has surprisingly been discovered that mice given the lectins pokeweedmitogen chloroform precipitatable fraction and Momordica lectinexhibited a significant decline in circulating glucose levels that wasnot secondary to the decline in food intake that also developed, or tochanges in insulin secretion produced by the lectin. Furthermore, thedecline in food intake which developed alone was not sufficient to causethe rapid weight loss that resulted in mice given the pokeweed mitogenand Momordica lectins. Instead, this was the combined result of thedecline in food intake and concomitant hypoglycemia.

Essentially any lectin capable of inducing both hypoglycemia and adecline in food intake is suitable for use in the present invention.Suitable lectins may be identified by administering candidate lectins tolaboratory test animals and then observing the test animals for aninduced lowering of serum glucose levels by a statistically significantdegree accompanied by a statistically significant weight loss.

Examples of lectins suitable for use with the present invention include,but are not limited to, the pokeweed mitogen chloroform precipitatablefraction and the Momordica lectin. The chloroform precipitatablepokeweed mitogen fraction can be administered in the form of pokeweedmitogen containing other fractions, provided that the chloroformprecipitatable fraction is present. It is preferred, however, that thechloroform precipitatable fraction be administered essentially free oflectins Pa-2 to Pa-4. Unlike other components of pokeweed, the lectinsare not toxic.

The term “pokeweed mitogen” as used herein refers to the mixture of fivelectins obtained from the plant Phytolacca americana. Specific pokeweedmitogen lectins are individually referred to as Pa-1, Pa-2, Pa-3, andthe like. When administered as a mixture of the five lectins, eitherorally or by intra peritoneal injection, pokeweed mitogen inducedhypoglycemia, a decline in food intake, and concomitant weight loss, innormal mice, diabetic mice, mice without T or B lymphocytes, and obesemice.

According to this invention, in the treatment of adult onset diabetes,mammals are administered an effective amount of a lectin capable ofstimulating a decline in food intake and inducing hypoglycemia. Fromabout 1.0 to about 5.0 mg of lectin per kilogram of body weight willproduce the desired result; however, from about 1 to about 100micrograms of lectin per kilogram of body weight per day is preferablyadministered, and from about 25 to about 75 micrograms of lectin perkilogram of body weight per day is more preferably administered.

To induce weight loss, mammals are administered an effective amount of alectin capable of inducing a decline in food intake. From about 0.25 toabout 3.0 mg of lectin per kilogram of body weight is effective; howevera dosage amount 1 and about 100 micrograms per kilogram of body weightper day is preferred. Again, the lectin should be administered daily atthe beginning to maintain the decline in food intake. Most preferably,the mammal should be administered an amount of lectin effective toinduce a decline in food intake without inducing hypoglycemia.Typically, this is between about 25 to about 100 micrograms of lectinper kilogram of body weight per day.

The amount, frequency and period of lectin administration will varydepending upon factors such as the level of circulating blood glucoseand the weight of the patient. Usually, the administration of the lectinwill be daily initially and then less frequently once the desiredglucose level and body weight is obtained. Daily dosages may be given byrepeatedly administering a unit dosage form. In the alternative, acontinuous dosage form such as a timed-release tablet or capsule or atransdermal drug delivery device may be employed to continuouslyadminister the lectin.

To induce weight loss, the lectin should be administered daily until aweight loss of at least 10 percent, and preferably at least 20 percent,if attained, after which the dosage amount may be reduced oradministered less frequently. In treatment of adult-onset diabetes, thelectin should be administered daily until the circulating blood glucoselevel drops below about 150 mg/dl, and preferably below about 120 mg/dl,or until a weight loss of at least about 10 percent, and preferablyabout 20 percent is obtained, after which the lectin dosage may bereduced or administered less frequently. Preferably, the lectin dosageis not reduced or administered less frequently until both a reduction incirculating blood glucose level below about 150 mg/dl and a loss of bodyweight of at least about 10 percent is obtained. Precise dosage amountsand frequency, and subsequent changes thereof, may be determined duringinitial and subsequent screenings of circulating glucose levels and bodyweight, and is well within the ability level of the ordinarily skilledmedical practitioner.

The role of the triterpene compounds found in pokeweed in inducinghypoglycemia and weight loss has been ruled out. More particularly, thetriterpenes contained in ginseng stimulate glucose uptake in sheep redblood cells. Although this effect could have accounted for thehypoglycemia and weight loss that developed after pokeweed mitogen wasgiven to mice, the decline in glucose was unaffected when pokeweedmitogen was added to blood. Further, a substance extracted from apokeweed mitogen preparation by chloroform caused hyperglycemia, ratherthan hypoglycemia. It is believed that the triterpenes present inpokeweed may have binding properties similar to that of ginseng, but areantagonists rather than agonists. That is, they block the activity thatginseng-derived triterpenes stimulate.

A role for the T or B lymphocytes induced by pokeweed mitogen ininducing hypoglycemia and weight loss has also been ruled out by the useof Severe Combined Immuno Deficiency (SCID) mice. These animals lackfunctional T and B lympho-cytes, but responded with hypoglycemia andweight loss anyway, and also exhibited an increase in circulating IL-6.Blood IL-6 concentrations declined in mice given the TNF-α antagonist,suggesting that TNF-α synthesis was stimulated by pokeweed mitogen,though secretion was not. Circulating TNF-α was undetectable. Thepersistence of an IL-6 response despite blockade of TNF-α suggested thatpokeweed mitogen induced the direct release of this cytokine. IL-6 is amediator of hypoglycemia, and is believed to be contributor to thechanges induced by pokeweed mitogen.

The hypoglycemia following lectin administration was shown not to besecondary to an increase in circulating insulin. In fact, serumconcentrations of insulin declined after pokeweed mitogen was given.Mice made diabetic by treating with streptozotocin also had lowerglucose concentrations following pokeweed mitogen treatment, furtherruling out the involvement of insulin. Because neither a whollycytokine-mediated effect nor insulin involvement in the decline incirculating glucose could be demonstrated, the direct effect of pokeweedmitogen was indicated. Elevated deoxyglucose uptake by C2C 12 myotubesexposed to a Pa-1 enriched fraction of pokeweed mitogen suggests adirect mechanism for the hypoglycemia induced by pokeweed mitogen.Because insulin induced a decline in food intake and body weight when itwas infused into the brain, a direct mechanism is also indicated for theweight loss induced by pokeweed mitogen. The decline in body weightobtained following pokeweed mitogen administration was found to be dueto a loss of both protein and fat stores. The combination of a declinein food intake and hypoglycemia is thus believed to promote exaggeratedmechanisms of adaptation to starvation, such as elevated mobilization ofenergy from muscle and fat depots.

Potential toxicity of pokeweed mitogen as the cause of the metabolicchanges has been ruled out. Lectins from pokeweed have been shown to benon-toxic when given to mice and after ingestion by humans. Kidney,liver and lung function are not affected by pokeweed mitogen. Studies ofthe central nervous and cardiovascular systems in animals and humansexposed to pokeweed mitogen have also found no deleterious effects.Mortality resulting from higher chronic doses given to laboratoryanimals in studies in accordance with the present invention was believedto be secondary to starvation.

Administration of the dose can be oral, intravenous, parenteral,subcutaneous, intramuscular, transdermal, or by any other acceptablemethod. The lectins can be administered in any number of conventionaldosage forms. Oral preparations may be in the form of tablets orcapsules prepared using appropriate binders and excipients. Intravenous,parenteral, subcutaneous and intramuscular preparations include sterilesolutions or suspensions. Continuous dosage forms such as sustainedrelease oral preparations and devices for transdermal delivery are alsocontemplated. The solutions to be administered may be reconstitutedlyophilized powders and they may additionally contain preservatives,buffers, dispersants, and the like. The pharmaceutical compositionscontemplated by the above dosage forms can be prepared with conventionalpharmaceutically acceptable ingredients, using conventional formulationsand techniques.

Preferably, the lectin is administered either orally or, in thealternative, systemically via subcutaneous or intramuscular injection orby transdermal delivery. When administered orally to mice, pokeweedmitogen induced hypoglycemia, a decline in food intake, and concomitantweight loss in half of the mice in the experimental group. The failureof the other half to respond is not presently understood. However, 100percent of mice administered pokeweed mitogen systemically exhibitedanorexia and experienced a statistically significant drop in circulatingblood glucose levels and body weight. Thus, in circumstances whereinthere is no response to oral lectin administration, the lectin should beadministered systemically instead via intravenous, parenteral,subcutaneous, transdermal or intramuscular dosage methods.

The following examples further illustrate the present invention, and arenot to be construed as limiting the scope thereof. All parts andpercentages are by weight unless expressly indicated to be otherwise.

EXAMPLES

The effect of lectins on circulating blood glucose levels and bodyweight can be determined using standard techniques according to thefollowing test protocol.

Materials and Methods

Animals: Male BALB/c, C57BL/6, ob/ob and CB6 F1 (C57BL/6 x BALB/c) (fromJackson Labs, Inc., Bar Harbor, Me.) or BALB/c SCID (severe combinedimmune deficiency, from Taconic Farns, Germantown, N.Y.) mice werehoused in a 12/12 light/dark cycle with standard laboratory chow (PurinaMills, Inc., Ill.) and water ad lib for at least two days before use.

CB6 mice were made diabetic by injecting streptozotocin (200 mg/kg i.p.in PBS) one time and measuring blood glucose four days later. At thistime, the mice given streptozotocin had serum glucose concentrations of420±10 mg/dl, twice normal values.

Blood was collected by retro-orbital bleeding to determine diabeticstate prior to receiving pokeweed mitogen and to measure TNFα and IL-6concentrations one and three hours, respectively, after pokeweed mitogentreatment in the corresponding studies. For other measurements, blood,by cardiac puncture, and tissue samples were collected after sacrificeby cervical dislocation.

Reagents: Pokeweed mitogen (PKW), Momordica lectin, streptozotocin andTriton X-100 were purchased from Sigma Chemical Co. (St. Louis, Mo.) andchloroform from J. T. Baker, Inc. (Phillipsburg, N.J.). Serum glucosewas measured using a commercial kit (Sigma), and insulin concentrationswere determined using a radioimmunoassay kit (ICN, Costa Mesa, Calif.).The BCA protein assay was purchased from Pierce (Rockford, Ill.). IL-6and TNF-α were measured using enzyme-linked immunosorbant assays (R&DSystems, Minneapolis, Minn. and Biosource International, Inc.,Camarillo, Calif., respectively). The TNF-α antagonist (TNFbp) was thekind gift of Amgen Boulder, Inc. (Boulder, Colo.). TNFbp consists of theextracellular domains of two type 1 (p55) TNFα receptors linked topolyethylene glycol. The anti-IL′6 antibody (20F3) was grown in nudemice from hybridoma cells and purified using standard antibody isolationprocedures (Pierce, Rockford, Ill.). For the glucose uptake study,2-deoxy-D-[1-³H]glucose was purchased from Amersham Life Science (ElkGrove, Ill.). Dulbecco's modified eagle medium, RPMI and fetal bovineserum were purchased from JRH Biosciences (Lenexa, Kans.). Horse serumwas purchased from Sigma. For T and B lymphocyte isolation, the Ficollwas from Sigma, the magnetic beads were from Dynal (Lake Success, N.Y.),the antibodies were from Becton-Dickinson (Mountain View, Calif.). The³H-thymidine was from New England Nuclear (Boston, Mass.).

Treatments: Pokeweed mitogen was dissolved in sterile PBS and given byintra peritoneal injection (i.p.), or orally, by gavage, in a volume of200 μl/20 g mouse. For dosages of 15 mg/kg, 300 μg PKW was dissolved in200 μl water. For 10 mg/kg and 5 mg/kg dosages, the level of PKW wasreduced proportionately. TNFbp and anti-IL-6 were given i.p. two hoursprior to PKW in the studies described. The TNFbp dose was 780 μg/mousein 100 μl of vehicle. The quantity of TNF-α antagonist given was 35-foldgreater than a dose which protected against death in theD-galactosamine/LPS model of liver injury disclosed by Russell et al.,J. Infect. Dis., 171, 1578-83(1995). The dosage of anti-IL-6 (20F3) was650 μg/mouse, in a 150 μl volume. Control animals received the samevehicle and volume at the appropriate time.

Chloroform extraction of pokeweed mitogen: The PKW solution used totreat mice was mixed thoroughly with an equal volume of chloroform byvortexing at moderate speed for 30 minutes. A precipitate appeared inthe chloroform layer which was separated by centrifugation. Both theclear chloroform layer and the precipitate were dried under nitrogen atroom temperature and then under vacuum to remove the chloroform. Theseextracts were redissolved in the same volume of the original vehicle(PBS). Mice were treated i.p. with the extracted pokeweed solution orwith the reconstituted extracts in the subsequent study reported below.

The chloroform layer contained no protein, while the precipitatecontained 14 percent as much protein as the unextracted PKW solution.The protein content of the aqueous layer plus the precipitate was equalto the protein content of the unextracted solution.

Human B and T lymphocyte stimulation by chloroform-treated pokeweedmitogen: B cells and T lymphocytes were purified from heparinized humanblood by separating PBMC over a Ficoll gradient, followed by passageover a nylon wool column. Adherent cells were depleted of non-Blymphocytes by using dynal magnetic beads either directly conjugatedwith anti-CD4 and anti-CD8 antibody, or by adding anti-CD16 or -CD14antibody to the cells and following this with anti-mouse IgG orIgM-coated beads in a secondary step. The B lymphocytes were plated as1×10⁵ cells/well and the T lymphocytes were plated as 2×10⁵ cells/wellon a 96 well plate. The three pokeweed mitogen fractions—unextracted,extracted and chloroform precipitate—were added and, two days later, 0.5μCi ³H-thymidine was added to each well. At the end of eight hours,thymidine uptake into the cells was measured.

Ex vivo exposure of blood to pokeweed mitogen: 75 μg of the pokeweedmitogen mixture in a volume of 50 μg was added to 0.5 mL of heparinizedblood. This is equal to the contribution that the PKW solution used foracute mouse studies would give to 2 mL of blood, roughly the bloodcontent of a 20 g mouse. The treated blood was incubated for six hoursat 38° C. in 5 percent CO₂. Blood was removed at intervals, plasma wasseparated and glucose was measured.

Deoxyglucose uptake in vitro: Murine-derived C2C12 myoblast cells wereobtained from the American Type Culture Collection (Rockville, Md.).These cells were plated in 24 well plates and grown in high glucose DMEMcontaining 10 percent fetal bovine serum. At confluence, the medium wasreplaced with high glucose RPMI containing 10 percent horse serum. Fourdays later, myotubes were formed, and the medium was replaced with lowglucose DMEM+2 percent FBS containing the treatments. Sixteen hourslater, 0.2 μCi 2-deoxy-D-[1-³H]glucose/well was added for 20 minutes.Afterwards, the cells were washed three times with PBS, solubilized in 1percent Triton X-100 and radioactivity and protein concentrations weremeasured.

Statistics: In all groups, n=3-10. The data are presented as mean±SEM.Groups were compared using the two tailed paired or unpaired t-test ortwo-way ANOVA, followed by the post hoc Fisher's test, as appropriate.When comparing body weights, except where noted (Table I, study II), allgroups which were statistically different from the control group at thetime of sampling also differed significantly when compared to theirinitial (pretreatment) body weights.

Results

Circulating glucose concentrations: Glucose concentrations in thevehicle-treated control group were 154±3 mg/dl. Mice given 15 mg/kg PKWshowed a significant hypoglycemia by four hours, declining to 78±11mg/dl, p<0.05 vs. controls. These concentrations declined further, to23±10 mg/dl at 24 hours (FIG. 1a). Blood lactate concentrations changedin parallel with glucose, being 39±4 mg/dl in PBS-treated animals, 27±4mg/dl four hours later and 16±2 mg/dl at 24 hours.

The hypoglycemia that followed PKW administration was not secondary tostimulated insulin release. Plasma insulin concentrations decreasedafter receiving PKW, from 43±10 μU/mL to 24±0 μU/mL 24 hours later,p<0.05 (FIG. 1b), while diabetic mice given PKW had a circulatingglucose 45 percent lower than the diabetic control group, also p<0.05(Table I). A decline in food intake did not contribute significantly tothe lowered glucose levels in mice given PKW (Table I).

The effects of pokeweed mitogen were induced by oral administration (bygavage) in 50 percent of the animals that received it. Thenon-responders had blood glucose of 210±17 mg/dl, while the respondershad blood glucose of 70±5 mg/dl and significantly lower body weights(Table I).

TABLE I Circulating Glucose, Food Intake and Weight Loss Induced ByVarious Treatments in Normal, SCID, and Diabetic Mice Glucose, Foodintake, Body Treatment mg/dl g/mouse Weight, g I. vehicle 248 ± 13  3.2617.5 ± 0.3 PKW only 34 ± 7* 0.07 17.0 ± 0.3 PKW + anti-IL-6 body  81 ±22*⋄ 0.22 17.7 ± 0.3 PKW + TNFbp  112 ± 19*⋄ 0.93 17.3 ± 0.4 PKW +anti-IL-6 + TNFbp  120 ± 14*⋄ 0.16 18.5 ± 0.5 II. vehicle 191 ± 6  3.5518.8 ± 0.2 PKW  74 ± 12* 0  16.3 ± 0.6* chloroform-extracted PKW 144 ±19  0.38  16.2 ± 0.3* chloroform extract 265 ± 24* 2.63 17.1 ± 0.7chloroform precipitate 106 ± 14* 0  16.0 ± 0.2* III. SCID + vehicle 126± 8  2.85 —∇ SCID + PKW 77 ± 8* 0.60 — IV. diabetes + vehicle 421 ± 14 — 16.0 ± 0.3 diabetes + PKW 227 ± 40* —  13.2 ± 0.8* V. vehicle 205 ±17  — — PKW 96 ± 5* — — fasting 192 ± 12  — — VI. oral, non-responder210 ± 17  — 18.6 ± 0.2 oral responder 70 ± 5* —  16.5 ± 0.2* *p < .05vs. control group in respective study. ⋄p < .05 vs. respectivePKW-treated group. ♦NS vs. initial body weight. ∇not done.

Body composition and food intake: A single treatment of 15 mg/kg PKW hadeffects lasting for three days (FIG. 2a). The decline in body weight atthis time was due to the loss of both lean tissue and adipose tissue.Lean tissue, represented by carcass weight, was 14.9±0.4 g in thecontrol group and 12.8±0.4 g in the PKW-treated group, p<0.05. Theadipose tissue, represented by the epididymal fat pad, was 0.12±0.01 gin the control group and 0.07±0.01 g in the PKW-treated group, p<0.05.These tissue comparisons are illustrated in FIG. 2b.

Chronic treatment with PKW: When mice were given PKW at a dose of 5mg/kg i.p. once every two days, body weight and food intake declineddramatically. On day three, after two treatments, three of the five micewere dead.

In a second part of this study, illustrated in FIGS. 3a and 3 b, micewere given PKW at a lower dose, 3 mg/kg/2 days, for 16 days. Food intakerecovered one day after each treatment. Beyond three days, the averagefood intake was roughly equal to that of the control group, oscillatingabove and below it in response to PKW. In the treated group, body weightwas 21.0±0.2 g at the start, but declined 15 percent, to 17.9±0.4 g byday five (p<0.05 vs. both Day 0 and the control group). The body weightof this group increased after day five, but remained below that of thecontrol group for the additional ten days of treatment and for one weekof monitoring after the treatment was discontinued.

Immune mediation of hypoglycemia: Circulating TNFα did not increaseafter a 15 mg/kg dose of PKW was given. However, an elevation in thecirculating concentration of IL-6, to 129±6 ng/mL, was found, comparedto undetectable levels in control animals (Table II). Pretreatment withTNFbp attenuated the IL-6 appearance 32 percent. The blood glucoseconcentration was significantly higher in mice given PKW+TNFbp, as wasfood intake. Despite an effect, both these indices remained low (TableI).

Mice given anti-IL-6 antibody prior to 15 mg/kg i.p. PKW did not presentwith a decline in circulating IL-6 concentrations (Table II). AlthoughIL-6 concentrations appeared unaffected, the anti-IL-6 attenuatedhypoglycemia to an extent which was equivalent to the effect of TNFbp,but anorexia was unaffected, shown in Table I. The cytokine antagonistswere not additive in their protective effects. All of the groups in thisspecific study lost weight overnight, possibly due to the stress ofbeing given two i.p. injections and eyebled in the span of three or fivehours.

SCID mice, which do not have functional T or B lymphocytes and cannotmount an allergic reaction, were not less sensitive to the hypoglycemiaeffect of PKW (Table I). These immunodeficient mice also responded withthe appearance of circulating IL-6, to 92±35 ng/mL in the treatedanimals (Table II).

TABLE II Cytokine Appearance After PKW Administration TNFα pg/mLIL-6.ng/mL I. Normal vehicle not detectable not detectable PKW only notdetectable 129 ± 6* PKW + anti-IL-6  —⋄ 123 ± 22* antibody PKW + TNFbpnot detectable  40 ± 5* PKW + anti-IL-6 + TNFbp — 104 ± 5* II. SCIDvehicle — not detectable PKW —  92 ± 35* *p < .05 vs. control group inrespective study. ⋄not done.

Chloroform extraction of pokeweed mitogen: Data from this study arepresented in Table I. The vehicle-treated group had circulating glucoseconcentrations of 19±16 mg/dl, while in the PKW-treated group, glucoselevels were 74±12 mg/dl, p<0.05. The same solution of pokeweed mitogenwas mixed with chloroform, the results described below.

The aqueous chloroform-extracted PKW solution had only a mildhypoglycemia effect, to 144±19 mg/dl, NS. Food intake declined, but bodyweight did not.

The chloroform precipitate induced a significant hypoglycemia (106±14mg/dl), weight loss and complete anorexia. This was in spite of a lowerprotein content in the reconstituted precipitate solution, constitutinga dosage of 2.1 mg protein/kg, rather than 15 mg/kg.

The chloroform extract, which did not contain any protein, had no effecton body weight or food intake. With this preparation, the circulatingglucose concentrations increased to 265±24 mg/dl, p<0.05 vs. controls.

Human B cell and T lymphocyte stimulation by pokeweed mitogens:Unstimulated thymidine uptake by B lymphocytes was 241±4 cpm/well. At 5μg/mL, the chloroform precipitate increased thymidine uptake by B cellsto 2633±267 cpm/well, 40 percent greater than unextracted PKW and 265percent greater than the chloroform-extracted solution of PKW. RegardingT lymphocytes, baseline thymidine uptake was 750±46 cpm/well. Theextracted PKW caused the greatest thymidine uptake, to 42213±762cpm/well. This was 24 percent greater than the PKW solution and 39percent greater than the chloroform precipitate (Table IV).

Ex vivo exposure of blood to pokeweed mitogen: Incubating mouse bloodwith PKW had no effect on the rate of glucose disappearance in the blood(Table III).

TABLE III Blood Glucose Concentrations After Vehicle Or Pokeweed MitogenAdministration For Various Incubation Periods Hour Vehicle PokeweedMitogen 0 206 ± 7   196 ± 12 1 142 ± 10 142 ± 9 2 111 ± 10 111 ± 9 3 92± 8  87 ± 4 6  9 ± 7  7 ± 4

Glucose concentrations are mg/dl of plasma. PKW was added at a level of150 μg/mL of blood.

TABLE IV B Lymphocyte And T Lymphocyte Stimulation Of By PokeweedMitogen And Chloroform Fractions Of Pokeweed Mitogen B Lymphocyte TLymphocyte vehicle  241 ± 4  750 ± 46 pokeweed mitogen 1876 ± 291* 36696± 1001 chloroform extracted  720 ± 10 42243 ± 767* PKW chloroform 2633 ±267* 33359 ± 1457 precipitate *p < .05 vs. vehicle and chloroformextracted PKW

The protein administration was 5 μg/mL for all treated groups,incubations for three days. Exposure to 0.5 μCi ³H-thymidine was foreight hours. Measurements represent cellular ³H-thymidine uptake ascpm/well.

Deoxyglucose uptake by C2C12 myotubes: The baseline glucose uptake inthese cells was 63±7 cpm/mg cellular protein. An insulin concentrationof 1 μM stimulated a 48 percent increase in deoxyglucose uptake, while10 μg/mL of the chloroform precipitate fraction of PKW stimulateddeoxyglucose uptake a similar amount, to 93±12 cpm/mg protein (bothp<0.05). The stimulation of deoxyglucose uptake by the chloroformprecipitate fraction of PKW was confirmed in a second study comparingthe PKW fractions, shown in Table V.

TABLE V ³H-deoxyglucose Uptake by C2C12 Myotubes Incubated WithFractions of Pokeweed Mitogen cpm/mg Cellular Protein vehicle 44.3 ± 3.9chloroform precipitate  61.3 ± 4.9* chloroform-extracted 54.9 ± 5.9pokeweed mitogen pokeweed mitogen 44.2 ± 3.8 *p < .05 vs. vehicle

20 μl of PBS or the treatment solutions were added to each well,yielding 4 μg/mL of the precipitate, 26 μg/mL of the extracted solutionand 30 μg/mL of the pokeweed mitogen solution used to derive the othertreatments. The myotubes were incubated with the treatments for 18 hoursprior to ³H-deoxyglucose exposure. Incubation with 0.2 μCi³H-deoxyglucose was for 20 minutes; and n=8-12 wells/group.

Treatment of obese mice with PKW: When obese ob/ob mice were given asingle PKW dose (10 mg/kg i.p.), serum glucose levels and food intakedeclined dramatically. On day two, half of one group of mice died,likely due to starvation. This is attributed to their abnormalmetabolism. Apparently obese ob/ob mice are more sensitive to PKW. Theremaining group, n=4, was used for the data depicted in Table 6. Themice were given the PKW and blood was collected just prior toadministration by retro-orbital bleeding and 18 hours later by cardiacpuncture.

TABLE VI Food Intake, ob/ob Mice g/Mouse Serum Glucose wt. Loss beforepokeweed mitogen 4.1 424 ± 4 after 10 mg/kg 0.0  55 ± 8 2%

This demonstrates that the obese ob/ob mouse, used as a model ofobesity, is sensitive to PKW. Although the mice did not lose as muchweight as normal mice, this was expected on the basis of theirdocumented lower metabolism and higher body fat content, i.e., the lostfat weighs less than protein or carbohydrate loss because of the waterassociated with the latter two body compartments.

Acute treatment with Momordica lectin: When mice were given a singleMomordica lectin dose (15 mg/kg i.p.), circulating blood glucose levels,body weight and food intake also declined dramatically, to a degreeequivalent to that experienced with PKW. The results are depicted inTable VII:

TABLE VII Effects Of Different Lectins On Circulating Glucose And BodyWeight In Mice Food Eaten Body Weight, g Weight Glucose Treatment Day 0Day 1 Change g/Mouse mg/dl vehicle 22.2 ± 1.0 22.5 ± 0.9   0.3 ± 0.21.56 223 ± 12 pokeweed 22.7 ± 0.6 20.7 ± 0.6 −1.9 ± 0.1 0.16 81 ± 7Momordica 22.4 ± 0.6 20.4 ± 0.2 −2.0 ± 0.5 0.20 124 ± 13

Pokeweed and Momordica lectins appear to have insulin-like activity inmice, and induce hypoglycemia and weight loss in these animals, evenwhen given orally. Such orally active proteins can be used in thetreatment of insulin-dependent diabetes mellitus or obesity, offering anew therapeutic strategy for treating these diseases.

The foregoing examples and description of the preferred embodimentshould be taken as illustrating, rather than as limiting, the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. All such modifications are intended to be included withinthe scope of the following claims.

What is claimed is:
 1. A pharmaceutical composition consistingessentially of an amount of a pokeweed mitogen chloroform precipitatablefraction effective to stimulate a decline in food intake, and apharmaceutically acceptable carrier.
 2. The pharmaceutical compositionof claim 1 in an oral dosage form.
 3. The pharmaceutical composition ofclaim 1 in a systemic dosage form.
 4. A pharmaceutical compositionconsisting essentially of an amount of a pokeweed mitogen chloroformprecipitatable fraction effective to induce hypoglycemia, and apharmaceutically acceptable carrier.
 5. The pharmaceutical compositionof claim 4 in an oral dosage form.
 6. The pharmaceutical composition ofclaim 4 in a systemic dosage form.